Table S1. PCR primers used in this work The primers were designed ...
Table S1. PCR primers used in this work Gene symbol MIF1
Usage cDNA qRT-PCR Localization (GFP) Localization (RFP) BiFc GST fusion in vitro translation
ZHD5
cDNA qRT-PCR Localization (GFP) BiFc GST fusion
5-F
in vitro translation
5-1
in vitro translation
5-2
in vitro translation
5-3
in vitro translation
5-4
in vitro translation
ATTA
EMSA
ATTA
DNA binding assay
eIF4A (At3g13920)
qRT-PCR
Polarity Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
Sequence 5’- AAAAAGCAGGCTTCATGATGAAGAAGAGACAGAT 5’- AGAAAGCTGGGTCAAGCGTTAGGTGGAGAATACT 5’- ACGGTTGCCGTGAGTTCAT 5’- AATTCCGGTGACAACCACAA 5’- AAAAAGCAGGCTTCATGATGAAGAAGAGACAGATG 5’- AGAAAGCTGGGTTTCAAGCGTTAGGTGGAGAATA 5’- GATCTCGAGATGATGAAGAAGAGACAGATG 5’- GGTGGATCCCGAGCGTTAGGTGGAGAATACTC 5’- GCTCAAGCTTCGATGATGAAGAAGAGACAGAT 5’- GGTGGATCCCGAGCGTTAGGTGGAGAATACTC 5’- AATAGGATCCATGATGAAGAAGAGACAGATGG 5’- ATCCGAATTCTCAAGCGTTAGGTGGAGAATAC 5’- CGCGCGATCGCATGATGAAGAAGAGACAGATG 5’- TCGTTTAAACTCAAGCGTTAGGTGGAGAATACTC 5’- AAAAAGCAGGCTATGGATATGAGAAGCCATG 5’- AGAAAGCTGGGTTTAGAGAGTAGTTGTTG 5’- GAAGTGTTCACGACGGATGC 5’- ACAAGCAGCGCATCTGAGAG 5’- AAAAAGCAGGCTTCATGGATATGAGAAGCCAT 5’- AGAAAGCTGGGTTTTAGAGAGTAGTTGTTGG 5’- TCGAATTCTATGGATATGAGAAGCCATGAAATG 5’- GGTGGATCCTTAGAGAGTAGTTGTTGGTGTTGG 5’- CAGGGATCCATGGATATGAGAAGCCAT 5’- ACGAATTCATTAGAGAGTAGTTGTTGG 5’- CGCGCGATCGCATGGATATGAGAAGCCAT 5’- TCGTTTAAACTTAGAGAGTAGTTGTTGG 5’- CGCGCGATCGCATGGATATGAGAAGCCAT 5’- TCGTTTAAACTTAGGTGGGTTTAGCTGCGGCGGA 5’- CGCGCGATCGCATGGTAAGATACCGTGAGTGT 5’- TCGTTTAAACCTAATCCGAGCTTCCAACGC 5’- CGCGCGATCGCATGGACGGCGTTGGAAGCTCG 5’- TCGTTTAAACTTAAGACGACGACATCGAAA 5’- CGCGCGATCGCATGAAGAAAAGATTCAGGACA 5’- TCGTTTAAACTTATGTTGGTGGTTTCTTAG 5’- CAGTGTCTTGTAATTAAAAA 5’- TTTTTAATTACAAGACACTG 5’- ATGGATCCGTAATTAAAAATTAAAAATTAAAAATTAAAAGCTTAT 5’- ATAAGCTTTTAATTTTTAATTTTTAATTTTTAATTACGGATCCAT 5’- TGACCACACAGTCTCTGCAA 5’- ACCAGGGAGACTTGTTGGAC
The primers were designed using the Primer3 software (version 0.4.0; 29). They had melting temperatures in a range of 50-60 oC. The underlined bases indicate restriction enzyme sites.
Table S2. Primers used for the construction of yeast plasmids Construct name AGI code ZHD1
At5g65410
Sequence
Restriction site
Function
5’- AAAATCGATGAATGGAGTTTGAAGACA
ClaI & XhoI
Prey
ClaI & XhoI
Prey
NdeI & XhoI
Prey
EcoRI & SacI
Prey
EcoRI & SacI
Prey
NdeI & XhoI
Prey
EcoRI & SacI
Prey
EcoRI & SacI
Prey
NdeI & SacI
Prey
NdeI & XhoI
Prey
EcoRI & SacI
Prey
NdeI & XhoI
Prey
NdeI & XhoI
Prey
EcoRI & SacI
Prey
NcoI & PstI
Bait
NcoI & PstI
Bait
NcoI & PstI
Bait
5’- AAACTCGAGTCATGGTTGGTCTTGTTC ZHD2
At4g24660
ZHD3
At2g02540
ZHD4
At1g14440
5’- AAAATCGATGAATGAATTTTGAGGATC 5’- AAACTCGAGTTAGGGTTTCTTACCAAG 5’- AAACATATGATGGAGATTGCAAGTCAAGA 5’- AAACTCGAGCTAAGGATTAGTAGAAGCAA 5’- ATAGAATTCATGGAAATTGCAAGTCAAGA 5’- ATTGAGCTCTCATGGGACGATCTTAGTC
ZHD5
At1g75240
5’- ATAGAATTCATGGATATGAGAAGCCATGA 5’- ATTGAGCTCTTAGAGAGTAGTTGTTGGTG
ZHD6
At2g18350
5’- AAAACATATGATGGAAGTTAGAGAGAAG 5’- AAACTCGAGTTACAAATCCTTCTTCTT
ZHD7
At3g50890
5’- ATAGAATTCATGGAGCTTGGAGGAAAATG 5’- ATTGAGCTCCTATTCACTAATGTTACTAT
ZHD8
At5g15210
5’- ATAGAATTCATGGATGTAATAGCTACTAC
ZHD9
At3g28920
5’- ATACATATGATGCTTGAAGTTAGATCAAT
ZHD10
At5g39760
5’- ATTGAGCTCTCACGTCGAAGAAAAC 5’- ATTGAGCTCTCACGACGAAGACGACGAG 5’- AAACATATGATGATGGATATGACTCCTAC 5’- AAACTCGAGTCACGACGACGATGATCCGT ZHD11
At1g69600
5’- ATAGAATTCATGGATTTGTCTTCCAAACC 5’- CCCGAGCTCTCAAGAAGATGAAGACCCA
ZHD12
At5g60480
5’- AAACATATGATGGTTGTCTTGTACAACGA 5’- AAACTCGAGCTACAACTTCAAATTCAAAG
ZHD13
At5g42780
5’- AAACATATGATGGATGAGATAAAACCAAA
ZHD14
At1g14687
5’- ATAGAATTCATGCAGAGTACTTGTGTCTA
MIF1
At1g74660
5’- AAACTCGAGTCATTCATCAATTATAATTTT 5’- ATTGAGCTCTTAGTGATAAAACTTTT 5’- AGGGCCATGGTCATGATGAAGAAGAGACAGATGG 5’- ATCCCTGCAGAATCAAGCGTTAGGTGGAGAATAC MIF2
At3g28917
5’- AAGTCCATGGGCATGAGGAAGCGTCAGGTGGTGT 5’- TTTTCTGCAGTAATTTCCAGTAGAAGGAGGTGAA
MIF3
At1g18835
5’- TTTACCATGGAGATGAAGAAGAGGCAAGTGGTGA 5’- ATCCTGCAGCATCAGTTGTTTGCATTCGGAGGAG
The primers had melting temperatures in a range of 50-60 oC. The underlined bases indicate restriction enzyme sites.
Supplemental Data Nuclear import and DNA binding of the ZHD5 transcription factor is modulated by a competitive peptide inhibitor in Arabidopsis Shin-Young Hong, Ok-Kyoung Kim, Sang-Gyu Kim, Moon-Sik Yang, and Chung-Mo Park
Figure S1
β-galactosidase activity
A 100 60 50 40
*
*
30
*
20
β-galactosidase activity
*
10 0
Gal4 BD-AD AD ZHD ZHD ZHD ZHD ZHD ZHD ZHD 3 5 7 8 10 11 13 MIF1 (Bait)
B 100 90 80 70 60 50 40 30 20 10 0
* *
*
*
*
*
Gal4 BD-AD AD ZHD ZHD ZHD ZHD ZHD ZHD ZHD 3 5 7 8 10 11 13
C β-galactosidase activity
*
MIF2 (Bait) 100 35 30 25 20 15 10 5 0
* *
* *
*
*
Gal4 BD-AD AD ZHD ZHD ZHD ZHD ZHD ZHD ZHD 3 5 7 8 10 11 13 MIF3 (Bait)
FIGURE S1. β-galactosidase assays of yeast cells coexpressing the MIF and ZHD proteins. The pGAD vector containing the GAL4 gene was a positive control. Negative control was the interaction between the prey vector (pGADT7, AD) and the bait vector (pGBKT7, BD). The ZHD and MIF gene sequences were fused to the GAL4 AD-coding sequence in the prey vector and the GAL4 BD-coding sequence in the bait vector, respectively. The β-galactosidase activities were determined using ONPG (onitrophenyl-β-D-galactopyraniside) as substrate (31). The β-galactosidase activities were expressed in Miller units. Three measurements were averaged. Bars indicate standard error of the mean (t-test, *P
Usage cDNA qRT-PCR Localization (GFP) Localization (RFP) BiFc GST fusion in vitro translation
ZHD5
cDNA qRT-PCR Localization (GFP) BiFc GST fusion
5-F
in vitro translation
5-1
in vitro translation
5-2
in vitro translation
5-3
in vitro translation
5-4
in vitro translation
ATTA
EMSA
ATTA
DNA binding assay
eIF4A (At3g13920)
qRT-PCR
Polarity Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse Forward Reverse
Sequence 5’- AAAAAGCAGGCTTCATGATGAAGAAGAGACAGAT 5’- AGAAAGCTGGGTCAAGCGTTAGGTGGAGAATACT 5’- ACGGTTGCCGTGAGTTCAT 5’- AATTCCGGTGACAACCACAA 5’- AAAAAGCAGGCTTCATGATGAAGAAGAGACAGATG 5’- AGAAAGCTGGGTTTCAAGCGTTAGGTGGAGAATA 5’- GATCTCGAGATGATGAAGAAGAGACAGATG 5’- GGTGGATCCCGAGCGTTAGGTGGAGAATACTC 5’- GCTCAAGCTTCGATGATGAAGAAGAGACAGAT 5’- GGTGGATCCCGAGCGTTAGGTGGAGAATACTC 5’- AATAGGATCCATGATGAAGAAGAGACAGATGG 5’- ATCCGAATTCTCAAGCGTTAGGTGGAGAATAC 5’- CGCGCGATCGCATGATGAAGAAGAGACAGATG 5’- TCGTTTAAACTCAAGCGTTAGGTGGAGAATACTC 5’- AAAAAGCAGGCTATGGATATGAGAAGCCATG 5’- AGAAAGCTGGGTTTAGAGAGTAGTTGTTG 5’- GAAGTGTTCACGACGGATGC 5’- ACAAGCAGCGCATCTGAGAG 5’- AAAAAGCAGGCTTCATGGATATGAGAAGCCAT 5’- AGAAAGCTGGGTTTTAGAGAGTAGTTGTTGG 5’- TCGAATTCTATGGATATGAGAAGCCATGAAATG 5’- GGTGGATCCTTAGAGAGTAGTTGTTGGTGTTGG 5’- CAGGGATCCATGGATATGAGAAGCCAT 5’- ACGAATTCATTAGAGAGTAGTTGTTGG 5’- CGCGCGATCGCATGGATATGAGAAGCCAT 5’- TCGTTTAAACTTAGAGAGTAGTTGTTGG 5’- CGCGCGATCGCATGGATATGAGAAGCCAT 5’- TCGTTTAAACTTAGGTGGGTTTAGCTGCGGCGGA 5’- CGCGCGATCGCATGGTAAGATACCGTGAGTGT 5’- TCGTTTAAACCTAATCCGAGCTTCCAACGC 5’- CGCGCGATCGCATGGACGGCGTTGGAAGCTCG 5’- TCGTTTAAACTTAAGACGACGACATCGAAA 5’- CGCGCGATCGCATGAAGAAAAGATTCAGGACA 5’- TCGTTTAAACTTATGTTGGTGGTTTCTTAG 5’- CAGTGTCTTGTAATTAAAAA 5’- TTTTTAATTACAAGACACTG 5’- ATGGATCCGTAATTAAAAATTAAAAATTAAAAATTAAAAGCTTAT 5’- ATAAGCTTTTAATTTTTAATTTTTAATTTTTAATTACGGATCCAT 5’- TGACCACACAGTCTCTGCAA 5’- ACCAGGGAGACTTGTTGGAC
The primers were designed using the Primer3 software (version 0.4.0; 29). They had melting temperatures in a range of 50-60 oC. The underlined bases indicate restriction enzyme sites.
Table S2. Primers used for the construction of yeast plasmids Construct name AGI code ZHD1
At5g65410
Sequence
Restriction site
Function
5’- AAAATCGATGAATGGAGTTTGAAGACA
ClaI & XhoI
Prey
ClaI & XhoI
Prey
NdeI & XhoI
Prey
EcoRI & SacI
Prey
EcoRI & SacI
Prey
NdeI & XhoI
Prey
EcoRI & SacI
Prey
EcoRI & SacI
Prey
NdeI & SacI
Prey
NdeI & XhoI
Prey
EcoRI & SacI
Prey
NdeI & XhoI
Prey
NdeI & XhoI
Prey
EcoRI & SacI
Prey
NcoI & PstI
Bait
NcoI & PstI
Bait
NcoI & PstI
Bait
5’- AAACTCGAGTCATGGTTGGTCTTGTTC ZHD2
At4g24660
ZHD3
At2g02540
ZHD4
At1g14440
5’- AAAATCGATGAATGAATTTTGAGGATC 5’- AAACTCGAGTTAGGGTTTCTTACCAAG 5’- AAACATATGATGGAGATTGCAAGTCAAGA 5’- AAACTCGAGCTAAGGATTAGTAGAAGCAA 5’- ATAGAATTCATGGAAATTGCAAGTCAAGA 5’- ATTGAGCTCTCATGGGACGATCTTAGTC
ZHD5
At1g75240
5’- ATAGAATTCATGGATATGAGAAGCCATGA 5’- ATTGAGCTCTTAGAGAGTAGTTGTTGGTG
ZHD6
At2g18350
5’- AAAACATATGATGGAAGTTAGAGAGAAG 5’- AAACTCGAGTTACAAATCCTTCTTCTT
ZHD7
At3g50890
5’- ATAGAATTCATGGAGCTTGGAGGAAAATG 5’- ATTGAGCTCCTATTCACTAATGTTACTAT
ZHD8
At5g15210
5’- ATAGAATTCATGGATGTAATAGCTACTAC
ZHD9
At3g28920
5’- ATACATATGATGCTTGAAGTTAGATCAAT
ZHD10
At5g39760
5’- ATTGAGCTCTCACGTCGAAGAAAAC 5’- ATTGAGCTCTCACGACGAAGACGACGAG 5’- AAACATATGATGATGGATATGACTCCTAC 5’- AAACTCGAGTCACGACGACGATGATCCGT ZHD11
At1g69600
5’- ATAGAATTCATGGATTTGTCTTCCAAACC 5’- CCCGAGCTCTCAAGAAGATGAAGACCCA
ZHD12
At5g60480
5’- AAACATATGATGGTTGTCTTGTACAACGA 5’- AAACTCGAGCTACAACTTCAAATTCAAAG
ZHD13
At5g42780
5’- AAACATATGATGGATGAGATAAAACCAAA
ZHD14
At1g14687
5’- ATAGAATTCATGCAGAGTACTTGTGTCTA
MIF1
At1g74660
5’- AAACTCGAGTCATTCATCAATTATAATTTT 5’- ATTGAGCTCTTAGTGATAAAACTTTT 5’- AGGGCCATGGTCATGATGAAGAAGAGACAGATGG 5’- ATCCCTGCAGAATCAAGCGTTAGGTGGAGAATAC MIF2
At3g28917
5’- AAGTCCATGGGCATGAGGAAGCGTCAGGTGGTGT 5’- TTTTCTGCAGTAATTTCCAGTAGAAGGAGGTGAA
MIF3
At1g18835
5’- TTTACCATGGAGATGAAGAAGAGGCAAGTGGTGA 5’- ATCCTGCAGCATCAGTTGTTTGCATTCGGAGGAG
The primers had melting temperatures in a range of 50-60 oC. The underlined bases indicate restriction enzyme sites.
Supplemental Data Nuclear import and DNA binding of the ZHD5 transcription factor is modulated by a competitive peptide inhibitor in Arabidopsis Shin-Young Hong, Ok-Kyoung Kim, Sang-Gyu Kim, Moon-Sik Yang, and Chung-Mo Park
Figure S1
β-galactosidase activity
A 100 60 50 40
*
*
30
*
20
β-galactosidase activity
*
10 0
Gal4 BD-AD AD ZHD ZHD ZHD ZHD ZHD ZHD ZHD 3 5 7 8 10 11 13 MIF1 (Bait)
B 100 90 80 70 60 50 40 30 20 10 0
* *
*
*
*
*
Gal4 BD-AD AD ZHD ZHD ZHD ZHD ZHD ZHD ZHD 3 5 7 8 10 11 13
C β-galactosidase activity
*
MIF2 (Bait) 100 35 30 25 20 15 10 5 0
* *
* *
*
*
Gal4 BD-AD AD ZHD ZHD ZHD ZHD ZHD ZHD ZHD 3 5 7 8 10 11 13 MIF3 (Bait)
FIGURE S1. β-galactosidase assays of yeast cells coexpressing the MIF and ZHD proteins. The pGAD vector containing the GAL4 gene was a positive control. Negative control was the interaction between the prey vector (pGADT7, AD) and the bait vector (pGBKT7, BD). The ZHD and MIF gene sequences were fused to the GAL4 AD-coding sequence in the prey vector and the GAL4 BD-coding sequence in the bait vector, respectively. The β-galactosidase activities were determined using ONPG (onitrophenyl-β-D-galactopyraniside) as substrate (31). The β-galactosidase activities were expressed in Miller units. Three measurements were averaged. Bars indicate standard error of the mean (t-test, *P
